Conventional antibodies are large multi-subunit protein complexes comprising at least four polypeptide chains, including two light chains and two heavy chains. The heavy and light chains of antibodies contain variable (V) regions, which bind antigen, and constant (C) regions, which provide structural support and effector functions. The antigen binding region comprises two separate domains, a heavy chain variable domain (VH) and a light chain variable domain (VL). Complementarity determining regions (CDRs), short amino acid sequences in the variable domains of an antibody, provide antigen specificity. The heavy and light chains of an antibody molecule each provide three CDRs (CDR1, CDR2 and CDR3), therefore there are six CDRs for each antibody that can come into contact with the antigen, resulting in the antigen specificity.
A typical antibody, such as an IgG molecule, has a molecular weight of approximately 150 kD. Therapeutic use can be limited due to the relatively large size of an antibody, which can restrict tissue penetration or epitope access.
A number of smaller antigen binding fragments of naturally occurring antibodies have been identified following protease digestion (for example, Fab, Fab′, and F(ab′)2). These antibody fragments have a molecular weight ranging from approximately 50 to 100 kD. Recombinant methods have been used to generate alternative antigen-binding fragments, termed single chain variable fragments (scFv), which consist of VL and VH joined by a synthetic peptide linker. A scFv molecule has a molecular weight of approximately 25-30 kD.
While the antigen binding unit of a naturally-occurring antibody in humans and most other mammals is generally known to be comprised of a pair of variable regions, camelid species express a large proportion of fully functional, highly specific antibodies that are devoid of light chain sequences. The camelid heavy chain antibodies exist as homodimers of a single heavy chain, dimerized via their constant regions (U.S. Pat. Nos. 5,840,526 and 6,838,254; and U.S. Patent Application Publication No. 2003-0088074). The variable domains of these camelid heavy chain antibodies, referred to as VHH domains, retain the ability, when isolated as fragments of the VH chain, to bind antigen with high specificity (Hamers-Casterman et al. Nature 363:446-448, 1993; Gahroudi et al. FEBS Lett. 414:521-526, 1997).
Antigen binding single VH domains, called domain antibodies (dAb), have also been identified from a library of murine VH genes amplified from genomic DNA of immunized mice (Ward et al. Nature 341:544-546, 1989). Human single immunoglobulin variable domain polypeptides capable of binding antigen with high affinity have also been described (see, for example, PCT Publication Nos. WO 2005/035572 and WO 2003/002609).
However, a need remains for very small antibodies that can specifically bind antigen. Such small molecules could provide increased epitope access, better tissue penetration and could be used for any diagnostic or therapeutic application that utilizes antibodies or their fragments.